The import route of ADP/ATP carrier into mitochondria separates from the general import pathway of cleavable preproteins at the trans side of the outer membrane

The ADP/ATP carrier (AAC) of the mitochondrial inner membrane is synthesized in the cytosol without a cleavable presequence. The preprotein preferentially binds to the mitochondrial surface receptor Tom70 and joins the import pathway of presequence-carrying preproteins at the cis side of the outer membrane. Little is known about the translocation of the AAC across the outer membrane and where its import route separates from that of cleavable preproteins. Here we have characterized a translocation intermediate of AAC during transfer across the outer membrane. The major portion of the preprotein is exposed to the intermembrane space, while a short segment is still accessible to externally added protease. This intermediate can be quantitatively chased to the fully imported form in the inner membrane. Its accumulation depends on Tom7, but not on the intermembrane space domain of Tom22 in contrast to cleavable preproteins. Moreover, opening of the intermembrane space inhibits the import of AAC, but not that of cleavable preproteins into mitoplasts. We conclude that the import route of AAC diverges from the general import pathway of cleavable preproteins already at the trans side of the outer membrane.     

Sorting of D-lactate dehydrogenase to the inner membrane of mitochondria. Analysis of topogenic signal and energetic requirements

D-Lactate dehydrogenase (D-LD) is located in the inner membrane of mitochondria. It spans the membrane once in an Nin-Cout orientation with the bulk of the protein residing as a folded domain in the intermembrane space. D-LD is synthesized as a precursor with an N-terminal cleavable presequence and is imported into the mitochondria in a Deltapsi-dependent, but mt-Hsp70-independent manner. Upon import in vitro D-LD folds in the intermembrane space to attain a conformation indistinguishable from endogenous D-LD. Sorting of D-LD to the inner membrane is directed by a composite topogenic signal consisting of the hydrophobic transmembrane segment and a cluster of charged amino acids C-terminal to it. We propose a model for the mode of operation of the sorting signal of D-LD. This model also accounts for the driving force of translocation across the outer membrane, in the apparent absence of mt-Hsp70-dependent assisted import and involves the folding of the D-LD in the intermembrane space.     

The sorting route of cytochrome b2 branches from the general mitochondrial import pathway at the preprotein translocase of the inner membrane

Cytochrome b2 is synthesized in the cytosol with a bipartite presequence. The first part of the presequence targets the protein to mitochondria and mediates translocation into the mitochondrial matrix compartment; the second part contains the sorting signal that is required for delivery of the protein to the intermembrane space. The localization of the structures that recognize the sorting signal is unclear. Here we show that upon import in vivo, the sorting signal of cytochrome b2 causes an early divergence from the general matrix import pathway and thereby prevents translocation of a folded C-terminal domain into mitochondria. By co-immunoprecipitations we find that translocation intermediates of cytochrome b2 are associated with Tim23, a component of the inner membrane protein import machinery. Cytochrome b2 constructs with an alteration in the sorting signal are mistargeted to the matrix of wild-type mitochondria. In mitochondria containing a mutant form of Tim23, however, the translocation of the altered sorting signal across the inner membrane is inhibited, and cytochrome b2 is correctly sorted to the intermembrane space. We suggest that the sorting signal of cytochrome b2 is recognized within the inner membrane in close vicinity to Tim23.     

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.     

Analysis of the sorting signals directing NADH-cytochrome b5 reductase to two locations within yeast mitochondria

Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space. We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations. The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space. A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space. Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively. These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems.     

Transport of proteins into the various subcompartments of mitochondria

The import of proteins into mitochondria is an intricate process comprised of multiple steps. The first step involves the sorting of cytosolically synthesized precursor proteins to the mitochondrial surface. There precursor proteins are recognized by specific receptors which deliver them to the general import site present in the outer membrane. The second stage of import involves a series of complex intraorganelle sorting events which results in the delivery of the proteins to one of the four possible submitochondrial destinations, namely the outer and inner membranes, the matrix and intermembrane space. Here in this review, we discuss the current knowledge on these intramitochondrial sorting events. We especially focus on targeting of proteins to the intermembrane space. Sorting to the intermembrane space represents a particularly interesting situation, as at least three separate targeting pathways to this subcompartment are known to exist.     

Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.     

The preprotein translocase of the mitochondrial inner membrane: function and evolution

Growing mitochondria acquire most of their proteins by the uptake of mitochondrial preproteins from the cytosol. To mediate this protein import, both mitochondrial membranes contain independent protein transport systems: the Tom machinery in the outer membrane and the Tim machinery in the inner membrane. Transport of proteins across the inner membrane and sorting to the different inner mitochondrial compartments is mediated by several protein complexes which have been identified in the past years. A complex containing the integral membrane proteins Tim17 and Tim23 constitutes the import channel for preproteins containing amino-terminal hydrophilic presequences. This complex is associated with Tim44 which serves as an adaptor protein for the binding of mtHsp70 to the membrane. mtHsp70, a 70 kDa heat shock protein of the mitochondrial matrix, drives the ATP-dependent import reaction of the processed preprotein after cleavage of the presequence. Preproteins containing internal targeting information are imported by a separate import machinery, which consists of the intermembrane-space proteins Tim9, Tim10, and Tim12, and the inner membrane proteins Tim22 and Tim54. The proteins Tim17, Tim22, and Tim23 have in common a similar topology in the membrane and a homologous amino acid sequence. Moreover, they show a sequence similarity to OEP16, a channel-forming amino acid transporter in the outer envelope of chloroplasts, and to LivH, a component of a prokaryotic amino acid permease, defining a new PRAT-family of preprotein and amino acid transporters.     

An import signal in the cytosolic domain of the Neurospora mitochondrial outer membrane protein TOM22

TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The protein is anchored to the lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The orientation of the protein is not influenced by the charges flanking its trans-membrane segment, in contrast to observations regarding proteins of other membranes. In vitro import studies utilizing TOM22 preproteins harboring deletions or mutations in the cytosolic domain revealed that the combination of the trans-membrane segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment of the cytosolic domain was found to be essential for the import and assembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in which the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 contains physically separate import and membrane anchor sequences.     

Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins

Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p co-purify in successive chromatographic fractionations and co-immunoprecipitated with each other. Tim9p can be cross-linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.     

Two distinct and independent mitochondrial targeting signals function in the sorting of an inner membrane protein, cytochrome c1

Proteins of the mitochondrial inner membrane display a wide variety of orientations, many spanning the membrane more than once. Some of these proteins are synthesized with NH2-terminal cleavable targeting sequences (presequences) whereas others are targeted to mitochondria via internal signals. Here we report that two distinct mitochondrial targeting signals can be present in precursors of inner membrane proteins, an NH2-terminal one and a second, internal one. Using cytochrome c1 as a model protein, we demonstrate that these two mitochondrial targeting signals operate independently of each other. The internal targeting signal, consisting of a transmembrane segment and a stretch of positively charged amino acid residues directly following it, initially directs the translocation of the preprotein into the intermembrane space. It then inserts into the inner membrane from the intermembrane space side in a delta psi-dependent manner and thereby determines the orientation the protein attains in the inner membrane. Analysis of a number of other presequence-containing protein of the inner membrane suggest that they too contain such internal targeting signals.     

Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5

Import of nuclear-encoded precursor proteins into mitochondria and their subsequent sorting into mitochondrial subcompartments is mediated by translocase enzymes in the mitochondrial outer and inner membranes. Precursor proteins carrying amino-terminal targeting signals are translocated into the matrix by the integral inner membrane proteins Tim23 and Tim17 in cooperation with Tim44 and mitochondrial Hsp70. We describe here the discovery of a new pathway for the transport of members of the mitochondrial carrier family and other inner membrane proteins that contain internal targeting signals. Two related proteins in the intermembrane space, Tim10/Mrs11 and Tim12/Mrs5, interact sequentially with these precursors and facilitate their translocation across the outer membrane, irrespective of the membrane potential. Tim10 and Tim12 are found in a complex with Tim22, which takes over the precursor and mediates its membrane-potential-dependent insertion into the inner membrane. This interaction of Tim10 and Tim12 with the precursors depends on the presence of divalent metal ions. Both proteins contain a zinc-finger-like motif with four cysteines and bind equimolar amounts of zinc ions.     

A GTP-dependent "push" is generally required for efficient protein translocation across the mitochondrial inner membrane into the matrix

Mitochondrial biogenesis requires translocation of numerous preproteins across both outer and inner membranes into the matrix of the organelle. This translocation process requires a membrane potential (DeltaPsi) and ATP. We have recently demonstrated that the efficient import of a urea-denatured preprotein into the matrix requires GTP hydrolysis (Sepuri, N. B. V., Schülke, N., and Pain, D. (1998) J. Biol. Chem. 273, 1420-1424). We now demonstrate that GTP is generally required for efficient import of various preproteins, both native and urea-denatured. The GTP participation is localized to a particular stage in the protein import process. In the presence of DeltaPsi but no added nucleoside triphosphates, the transmembrane movement of preproteins proceeds only to a point early in their translocation across the inner membrane. The completion of translocation into the matrix is independent of DeltaPsi but is dependent on a GTP-mediated "push." This push is likely mediated by a membrane-bound GTPase on the cis side of the inner membrane. This conclusion is based on two observations: (i) GTP does not readily cross the inner membrane barrier and hence, primarily acts outside the inner membrane to stimulate import, and (ii) the GTP-dependent stage of import does not require soluble constituents of the intermembrane space and can be observed in isolated mitoplasts. Efficient import into the matrix, however, is achieved only through the coordinated action of a cis GTP-dependent push and a trans ATP-dependent "pull."     

Preproteins of chloroplast envelope inner membrane contain targeting information for receptor-dependent import into fungal mitochondria

The amino-terminal transit sequences of two preproteins destined for the chloroplast inner envelope membrane show similarities to mitochondrial presequences in the prevalence of positive charges and the potential formation of an amphipathic alpha-helix. We studied if these preproteins could be imported into mitochondria and found a low, yet significant import into isolated plant mitochondria. The plant mitochondria were previously shown not to import precursors of chloroplast stromal or thylakoidal proteins. To analyze the specificity of import into mitochondria we used the established import systems of fungal mitochondria. The envelope preproteins were efficiently imported into Saccharomyces cerevisiae or Neurospora crassa mitochondria. Their import showed the characteristics of specific mitochondrial protein uptake, including a requirement for the main receptor MOM19 (mitochondrial outer membrane protein of 19 kDa) and a membrane potential across the inner membrane, and depended on the presence of the chloroplast transit sequence. We conclude that some chloroplast transit sequences contain sufficient information for specific interaction with mitochondrial import receptors (at least from fungal sources).     

The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.     

Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677-1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.     

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Tim44 driving system. By blue native electrophoresis, we identify an approximately 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.     

cis and trans sites of the TOM complex of mitochondria in unfolding and initial translocation of preproteins

Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. Our previous studies led to the concept of two preprotein binding sites acting in series, the surface-exposed cis site and the trans site exposed to the intermembrane space. We report here that preproteins are bound to the cis site in a labile fashion even at low ionic strength, whereas intermediates arrested at the trans site remained firmly bound at higher salt concentration. The stability of the trans site intermediate results from interactions of both the presequence and unfolded parts of the mature part of the preprotein with the TOM complex. Binding to the trans site proceeded at rates comparable with those of unfolding of the mature domain and appeared to be kinetically limited by the unfolding reaction. Efficient binding to the trans site and unfolding were observed with both outer membrane vesicles and intact mitochondria whose membrane potential, DeltaPsi, was dissipated. Upon re-establishing DeltaPsi, trans site-bound preprotein resumed translocation into the matrix. The rates of unfolding and binding to the trans site were the same as those for translocation into intact energized mitochondria. We conclude that preprotein unfolding in intact mitochondria can take place without the involvement of the translocation machinery of the inner membrane and, in particular, the matrix Hsp70 chaperone. Further, preprotein unfolding at the outer membrane can be a rate-limiting step for formation of the trans site intermediate and for the entire translocation reaction.     

Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria

Mitochondrial precursor proteins made in the cytosol bind to a hetero-oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This 'trans' domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N-terminal targeting sequence of a translocation-arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration-driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3- to 8-fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursor's basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.     

The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import

Import of preproteins into the mitochondrial matrix is driven by the ATP-dependent interaction of mt-Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit. We show that Mge1p, a co-chaperone of mt-Hsp70, plays a key role in the ATP-dependent import reaction cycle in yeast. Our data suggest a cycle in which the mt-Hsp70-Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP. Hydrolysis of ATP by mt-Hsp70 occurs in complex with Tim44. Mge1p is then required for the dissociation of the ADP form of mt-Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP. ATP hydrolysis and complex dissociation are accompanied by tight binding of mt-Hsp70 to the preprotein in transit. Subsequently, the release of mt-Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt-Hsp70. Rebinding of ATP to mt-Hsp70 completes the reaction cycle.